Single tube multiplex PCR method for the detection of HBA1, HBA2 and HBA12 genes

ABSTRACT

One aspect of the invention is a method for amplifying alpha globin genes HBA1, HBA2 and HBA12 in a single PCR tube to determine an HBA genotype of a subject. This method employs five primers selected to accurate and sensitively identify the HBA1, HBA2, and HBA12, a gene found at a higher frequency in citizens of Saudi Arabia, by accurately annealing to nucleic acids in a biological sample and simultaneously amplifying sequences encoding the alpha globin genes. This invention includes a procedure and required reagents for the amplification of alpha globin genes in a single PCR tube.

REFERENCE TO A SEQUENCE LISTING

In accordance with 37 CFR § 1.52(e)(5), the present specification makesreference to a Sequence Listing which is submitted herewithelectronically as a .txt file named “481382us_ST25.txt”. The .txt filewas generated on Nov. 12, 2018 and is 3,090 bytes in size. The entirecontents of the Sequence Listing are herein incorporated by reference.

BACKGROUND Field of the Invention

The invention pertains to the fields of medicine, genetics, andimmunology.

Description of Related Art

The “background” description provided herein is for the purpose ofgenerally presenting the context of the disclosure. Work of thepresently named inventor(s), to the extent it is described in thisbackground section, as well as aspects of the description which may nototherwise qualify as prior art at the time of filing, are neitherexpressly or impliedly admitted as prior art against the presentinvention.

HBA Genes. The HBA1 and HBA2 gene sequences encode alpha globinproteins. The HBA1 gene provides instructions for making a proteincalled alpha-globin and this protein is also produced from a nearlyidentical gene called HBA2. These two alpha-globin genes are locatedclose together in a region of chromosome 16 known as the alpha-globinlocus. They are homologous in nature and show frequent intergenicexchange; Michelson, A. M.; and Orkin, S. H.; (1983). Boundaries of geneconversion within the duplicated human alpha-globin genes. Concertedevolution by segmental recombination. Journal of Biological Chemistry.258(24): pp. 15245-15254; Law, H. Y.; Luo, H. Y.; Wang, W.; Ho, J. F.;Najmabadi, H. Ng. IS.; Steinberg, M. H.; Chui D. H.; Chong, S. S.(2006), Determining the cause of patchwork HBA1 and HBA2 genes:recurrent gene conversion or crossing over fixation events.Haematologica. 91(3):297-302; Hardison, R. C., (2012). Evolution ofhemoglobin and its genes. Cold Spring Harbor perspectives in medicine.2(12): p. a011627; Borgio et al., id. 2014; Borgio, id. 2015).

HBA1 and HBA2 as well as their mutant forms have a high degree ofmolecular similarity and gene conversion between HBA and HBA2 in thealpha globin locus region is well documented and recently it has beendiscovered that the HBA2 has been replaced by a unique HBA12 geneconversion in 5.7% of the Saudi population. Direct sequencing of theHBA2 and HBA1 genes from 157 Saudi subjects revealed a new HBA2 geneconversion in cis or trans in 5.7% of these subjects. This new HBA2 geneconvert is referred to as the α12 (HBA12) allele due to its combinationof α1 (HBA1) and a2 (HBA2) sequences; see Borgio, J. F.; AbdulAzeez, S.,Al-Nafie, A. N., Naserullah, Z. A., Al-Jarrash, S., A novel HBA2 geneconversion in cis or trans: “alpha12 allele” in a Saudi population,Blood Cells, Molecules and Diseases. 53: 199-203 (2014) and Borgio, J.F., Molecular nature of alpha-globin genes in the Saudi population,Saudi Medical Journal 36:1271-1276 (2015).

HBA12 Gene. The HBA12 gene allele comprises parts of the HBA1 gene(promoter, intron 1, coding region 2, intron 2) in its upstream region,while downstream of HBA12 gene is indistinguishable from the HBA2 gene(part of intron 2 and exon 3); Borgio et al., id. 2014, id.; Borgio, id.2015. The HBA12 gene has the region starting −6 bp until 581 bp (3′promoter, exon1, IVSI, exon2, and 5′IVSII) from HBA1 gene, and 774 bp(3′enhancer) onwards from HBA2 gene; and a total of 5.7% of the studypopulation including sickle cell trait, hemophilia-A patient, SCDpatients, and β-thal major patients were reported to have the new geneconvert, α₁₂ gene; Borgio, id. (2014).

Reduced HBA2 in HBA12 patients. A reduced level of HBA2 was observed inthe first five (HbS^(carrier); β-thal^(carrier);β-thal^(major)/α-thal^(carrier); SCD^(+ve), and α-thal^(carrier);HbS^(carrier) α-thal^(carrier)) of six different subgroups of Saudis whocarried HBA12; Borgio, et al., id. (2014). These groups as well as 32different genotypes reported in the Saudi population are incorporated byreference to Borgio, et al., id. (2014). The presence of HBA12 genotypesα1α12/α1α12, α1-/α1α12, α1α2/α1α12, −α23.7/α1α12, and α1-4.2/α1α12 isassociated with a reduced level of HBA2 (α262) in β-thalassemiacarriers, which might give a false negative result in conventionaltests; Borgio et al., id., 2014.

The HBA12 gene conversion presents challenges to conventional tests thatonly measure HBA1 or HBA2 and there is a need for a simple test that canbe used for large scale screening of populations, like the Saudipopulation, where HBA12 is prevalent in order to proper asses thedisease burden in these populations; Borgio, et al., id. (2015); Akhtar,M. S., Qaw, F., Borgio, J. F, Albuali, W., Suliman, A., Nasserullah, Z.,Al-Jarrash, S. and Al-Ali, A., Spectrum of α-thalassemia mutations intransfusion dependent β-thalassemia patients from the Eastern Provinceof Saudi Arabia. Hemoglobin. 37(1): 65-73 (2013).

Alpha-thalassemia (α-thal) is a disorder caused by the deletion ofsingle or double α-globin genes, and/or point mutations in the α-globingenes. The α-globin genes, such as HBA1 and HBA2 have been emerging as amolecular target for the treatment of β-thalassemia (β-thal), forexample, reduced synthesis of α-globin protein can ameliorate theclinical severity of β-thalassemia. Hence, it is essential to understandthe molecular nature of α-globin genes to treat the most prevalenthemoglobin disorders, such as sickle cell disease, α-thal, and β-thalprevalent in the Kingdom of Saudi Arabia. Many alpha globin genotypesfrom the Eastern Region of Saudi Arabia, such as α1α12/α1α12, α1-/α1α12,α1α2/α1α12, α23.7/α1α12, and α1-4.2/α1α12 have been identified, seeBorgio et al., id., 2014 and these genotypes have been observed insubjects having sickle cell disease (SCD), sickle cell trait,β-thalassemia major and in β-thal carriers; Borgio et al., id., 2014.These and other genotypes associated with these diseases areincorporated by reference to Borgio, et al., id. 2014) and Borgio, etal. id. 2015. The HBA12 genotypes α1α12/α1α12, α1-/α1α12, α1α2/α1α12,−α123.7/2, and α1-4.2/α1α12 have been associated with the level of HBA2and a high level of HBA2 is considered to be a marker for the presenceof the β-thalassemia mutation.

In view of the limitations of conventional techniques, especially withregard to Saudi patients having an HBA12 genotype, the inventors soughtto develop a multiplex PCR-based method that accurately identifies HBA12genotypes along with related alpha globin genotypes, such as the HBA1and HBA2 genotypes, as well as a test that avoids false positiveframeshift mutations due to deletion of a region containing 5′ CTCGGCCC3′ with an objective of diagnosing genetic diseases and disorders suchas thalassemia and sickle cell anemia as well as the associated carrierstates.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the invention is directed to a multiplex polymerasechain reaction (PCR) based method that simultaneously detects the HBA1,HBA2 and HBA12 genes in a biological sample. This method is particularlyuseful in geographic regions like Saudi Arabia where a significantpercentage of the population carry the HBA12 gene conversion or convert.This method simultaneously targets three different alpha globin genesHBA1, HBA2 and HBA12 in a biological a sample quickly, with highsensitivity, and at low cost. This avoids situations where conventionalPCR primers for the HBA1 and HBA2 genes cannot amplify the HBA12 genebecause the HBA12 gene results from patchwork between the HBA1 and HBA2genes. Only a single PCR procedure is required to provide diagnosisbased on detection of these three genes thus minimizing the cost andtime of conducing separate tests and the HBA1, HBA2, HBA12 multiplex PCRdescribed herein was engineered in such a way as to detect all thepossible combinations as described in FIG. 1D and FIG. 4. The method maybe used in hospitals, diagnostic laboratories, forensic laboratories,and research laboratories.

Additional, non-limiting embodiments of this technology include themethods and products described below.

One embodiment of the invention is method for determining an HBAgenotype and/or treating a subject at risk of a hemoglobin A (HBA)associated disease or trait comprising detecting Hemoglobin alpha 1(HBA1) gene, Hemoglobin alpha 2 (HBA2) gene, and Hemoglobin alpha 12(HBA12) genes in the subject, including: obtaining a sample from thesubject; contacting the sample with at least one set of primers for eachof HBA1, HBA2 and HBA12 under conditions suitable for the primers toamplify DNA in the sample by multiplex polymerase chain reaction (PCR),wherein the set of HBA1 primers are MA1F (SEQ ID NO: 3) and MA12R (SEQID NO: 4), the set of HBA2 primers are MA2F (SEQ ID NO: 5) and MA2R (SEQID NO: 2 or 6) and the set of HBA12 primers are MA12F (SEQ ID NO: 1) andMA2R (SEQ ID NO: 2 or 6), detecting presence of, or levels of, HBA1,HBA2 and HBA12 DNA amplified by each primer, thereby detecting agenotype of the subject, and, for a method of treatment, treating thesubject for an HBA associated disease or trait when a genotypeassociated with an HBA disease or trait is detected. In someembodiments, the subject carries gene conversion HBA12 and in otherembodiments subject does not carry gene conversion HBA12.

The method described in the embodiments above may be performed using abiological sample that is blood, plasma or serum, or white blood cells,or cells obtained from a buccal swab. In some embodiments a biologicalsample may be obtained from storage, such as from a bank of cell samplesor DNA.

The primers for HBA1, HBA2 and HBA12 may be part of a single primerpool.

The embodiments described above may further include selecting a subjectat risk of sickle cell disease when the presence of amplified HBA1, HBA2and/or HBA12 is detected; and/or may further include selecting orcharacterizing or treating a subject having sickle cell trait or sicklecell disease when the presence of amplified HBA1, HBA2 and/or HBA12 isdetected. In other related embodiments the method may further includeselecting a subject at risk of sickle cell disease when the presence ofamplified HBA1, HBA2 and/or HBA12 is detected and treating the subjectfor sickle cell disease with at least one of penicillin, hydroxyurea, atransfusion of red blood cells transfusion, by hematopoietic stem celltransplantation, by gene therapy of by other modes of treatmentdescribed herein.

In some embodiments a method disclosed herein includes selecting asubject at risk of thalassemia when the presence of amplified HBA1, HBA2and/or HBA12 is detected; and/or it may also include selecting a subjectat risk of thalassemia when the presence of amplified HBA1, HBA2 andHBA12 is detected and treating the subject for thalassemia with at leastone of a blood transfusion, iron chelation therapy, folic acid, or theother treatment modes described herein.

Another embodiment of the invention involves a primer pool compositioncomprising a set of primers for each of HBA1, HBA2 and HBA12, whereinthe set of HBA1 primers are MA1F (SEQ ID NO: 3) and MA12R (SEQ ID NO:4), the set of HBA2 primers are MA2F (SEQ ID NO: 5) and MA2R (SEQ ID NO:2 or 6) and the set of HBA12 primers are MA12F (SEQ ID NO: 1) and MA2R(SEQ ID NO: 2 or 6). A related embodiment involves a compositionincluding one or more human DNA samples or templates and the primer poolof embodiment 12, and optionally positive or negative control samplesfrom subjects not having a thalassemia or sickle cell disease or trait.Such a composition can also include human nucleic acid sample ortemplate, the primer pool of embodiment 11, a DNA polymerase, dNTPs, abuffer solution, and bivalent cations, monovalent cations.

Another embodiment of the invention is a pair or pairs of primers thatamplify at least one of HBA1, HBA2 and/or HBA12, selected from the groupconsisting of HBA1 primers MA1F (SEQ ID NO: 3) and MA12R (SEQ ID NO: 4);HBA2 primers MA2F (SEQ ID NO: 5) and MA2R (SEQ ID NO: 2 or 6); and HBA12primers MA12F (SEQ ID NO: 1) and MA2R (SEQ ID NO: 2 or 6). Amplificationis preferably performed in a single PCR tube will a combination of theseprimers, but in some embodiments may be performed sequentially or inparallel with individual primer pairs.

Related embodiments include a composition that includes HBA1 primersMA1F (SEQ ID NO: 3) and MA12R (SEQ ID NO: 4); HBA2 primers MA2F (SEQ IDNO: 5) and MA2R (SEQ ID NO: 2 or 6); and/or HBA12 primers MA12F (SEQ IDNO: 1) and MA2R (SEQ ID NO: 2 or 6).

In some embodiments, the method described above may be practiced with achemically-modified primer selected from the group consisting of MA1F(SEQ ID NO: 3) and MA12R (SEQ ID NO: 4), MA2F (SEQ ID NO: 5) and MA2R(SEQ ID NO: 2 or 6), MA12F (SEQ ID NO: 1), MA12aR (SEQ ID NO: 7), MA2SF(SEQ ID NO: 8), MA1R (SEQ ID NO: 9) or MA2SR (SEQ ID NO: 10); whereinsaid primer has been modified (a) by conjugation to a fluorescent tag,biotin, quencher or other detectable reporter moiety and/or bysubstitution of a chemically modified, non-natural nucleotide for atleast one dA, dC, dG or dT in MA1F (SEQ ID NO: 3) and MA12R (SEQ ID NO:4), MA2F (SEQ ID NO: 5) and MA2R (SEQ ID NO: 2 or 6), MA12F (SEQ ID NO:1), MA12aR (SEQ ID NO: 7), MA2SF (SEQ ID NO: 8), MA1R (SEQ ID NO: 9) orMA2SR (SEQ ID NO: 10. Other embodiments include compositions andindividual primer pairs including the chemically-modified forms of theprimers described herein.

Another embodiment is directed to premarital test or other genetic testkit comprising a set of primers for each of HBA1, HBA2 and HBA12,wherein the set of HBA1 primers are MA1F (SEQ ID NO: 3) and MA12R (SEQID NO: 4), the set of HBA2 primers are MA2F (SEQ ID NO: 5) and MA2R (SEQID NO: 2 or 6) and the set of HBA12 primers are MA12F (SEQ ID NO: 1) andMA2R (SEQ ID NO: 2 or 6), and, optionally, buccal swab(s), samplecontainers optionally containing preservatives for DNA, packagingmaterials, return mail or courier envelopes or containers, andinstructions for use for assessing a risk of sickle cell anemia, sicklecell trait, or thalassemia or thalassemia trait.

The foregoing paragraphs have been provided by way of generalintroduction, and are not intended to limit the scope of the followingclaims. The described embodiments, together with further advantages,will be best understood by reference to the following detaileddescription taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the disclosure and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIGS. 1A-1D. Primer sites and amplicon size for the multiplexamplification of the HBA1 (α1), HBA2 (α2) and HBA12 (α2) genes, seeFIGS. 1A, 1B and 1C, respectively. FIG. 1D describes a model multiplexPCR for the identification of α12 and its genotypes. The amplicons forFIGS. 1A, 1B and 1C are respectively shown by FIGS. 6, 7 and 8 and bySEQ ID NOS: 12, 13 and 14. As shown in FIG. 1D, lane 1, the normalα1α2/α1α2 genotype shows the α1 and α2 amplicons, but not the α12amplicon. Lanes 2-7 show patterns characteristics of the other genotypesdepicted at the top of the figure.

FIG. 2. Manually designed primers for amplification of HBA1, HBA2 andHBA12 genes.

FIG. 3A. Multiplex PCR results. Lane L: 100 bp Ladder; Lanes 1 to 11:Multiplex PCR result for various samples; and Lane AL: Allelic ladder.An 1874 bp amplicon (α2) is visible in lands 5, 6, 8-10 and AL. A 1231bp amplicon (α1) appears in lanes 1-11 and AL and a 396 bp amplicon (α2)appears in lanes 2, 11 and AL.

FIG. 3B. Multiplex PCR results. Lane L: 100 bp Ladder; Lane AL: Allelicladder showing the positions of each amplicon, respectively from top: α2(1874 bp), α1 (1231 bp) and α12 396 bp). Sample 1 and the Allelic laddershow the band corresponding to the α12 amplicon.

FIG. 3C. Multiplex PCR results-location of non-specific bands. Lanes X,Y and Z: Highlighted green color bands (between the upper band and thetwo lower bands) denote the non-specific PCR products. Lane AL: Allelicladder.

FIG. 4. Multiplex PCR and quick genotype reference guide: left and rightlanes L: 100 bp Ladder; Lane A: α1α2/α1α2; Lane B: α1α2/α1α12; Lane C:α1α12/α1α12; Lane C: α1α12/α1α12; Lane D: −α2/−α12; Lane E: α1-/α1-;Lane F: −α2/−α12; Lane G: −α2/−α2; Lane H: Allelic ladder.

FIG. 5. A sequence electropherogram from the HBA12 gene amplicon.Sequence at top is described by SEQ ID NO: 11.

FIG. 6. Nucleotide sequence of the PCR product from the primer pair MA2Fand MA2R from the HBA12 gene with the sequence length of 1874 bp long.This sequence appears as SEQ ID NO: 12.

FIG. 7. Nucleotide sequence of the PCR product from the primer pairMA12F and MA2R from the HBA12 gene with the sequence length of 396 bplong. This sequence appears as SEQ ID NO: 13.

FIG. 8. Nucleotide sequence of the PCR product from the primer pair MA1Fand MA12R from the HBA1 gene with the sequence length of 1231 bp long.This sequence appears as SEQ ID NO: 14.

DETAILED DESCRIPTION OF THE INVENTION

The multiplex PCR method disclosed herein may be used to identify orcharacterize the HBA genotype of a person, such as a patient or asubject at statistical risk of carrying an HBA12 gene conversion. Apatient or subject to be diagnosed, treated, or diagnosed and treatedwith a method disclosed herein may be a normal subject having a genotypenot associated with disease, a carrier or a subject carrying a diseasetrait, such as sickle cell trait, or a patient having a disease ordisorder associated with HBA1, HBA2 and/or HBA12. Genotypes such asthose described by FIG. 4 may be easily and inexpensively determined bythe multiple PCR method of the invention.

Hemoglobin genes. For practical purposes, the two alpha globin genes(termed alpha1 and alpha2) are identical. The HBA2 (α₂) and HBA1 (α₁)coding sequences are identical. These genes differ slightly over the 5′untranslated regions and the introns, but they differ significantly overthe 3′ untranslated regions. Since each normal cell has two chromosomes16, a total of four alpha globin genes exist in each cell. Each of thefour genes produces about one-quarter of the alpha globin chains neededfor hemoglobin synthesis.

Hemoglobin A1 also known as Hemoglobin A (HbA), adult hemoglobin, orα₂β₂, is the most common human hemoglobin tetramer, comprising over 97%of the total red blood cell hemoglobin. It consists of two alpha chainsand two beta chains; see Hemoglobinopathies, revised Apr. 17, 2002,hypertext transferprotocol://sickle.bwh.harvard.edu/hemoglobinopathy.html (last accessedOct. 30, 2018, incorporated by reference).

Hemoglobin A2 is a normal variant of hemoglobin A that consists of twoalpha and two delta chains (α₂δ₂) and is found at low levels in normalhuman blood. Hemoglobin A2 may be increased in beta thalassemia or inpeople who are heterozygous for the beta thalassemia gene. HBA2 existsin small amounts in all adult humans (1.5-3.1% of all hemoglobinmolecules) and is approximately normal in people with sickle-celldisease; Hemoglobinopathies, id. (2002).

Hemoglobin A12. The HBA12 gene comprises parts of the HBA1 gene(promoter, intron 1, coding region 2, intron 2) in its upstream region,while downstream of HBA12 gene is indistinguishable from the HBA2 gene(part of intron 2 and exon 3) (Borgio et al., id. 2014, id.; Borgio, id.2015). The intergenic exchange between the HBA1 and HBA2 genes wasobserved among 5.7% of Saudis (Borgio et al., id. 2014; Borgio, id.2015).

Thalassemia. The thalassemias are a group of disorders in which thenormal hemoglobin protein is produced in lower amounts than usual. Thegenes are defective in the amount of hemoglobin they produce, but thatwhich they produce (generally) is normal. The thalassemias are a complexgroup of disorders because of the genetics of hemoglobin production andthe structure of the hemoglobin molecule.

There are four kinds of alpha thalassemia: (i) Alpha thalassemia silentcarrier. One gene is missing or damaged, and the other 3 are normal.Blood tests are usually normal. A patient's red blood cells may besmaller than normal. Being a silent carrier means there are no signs ofthe disease, but one can pass the damaged gene on to one's offspring.This is confirmed by DNA tests. (ii) Alpha thalassemia carrier. Twogenes are missing. A subject may have mild anemia. (iii) Hemoglobin Hdisease. Three genes are missing. This leaves just 1 working gene. Apatient may have moderate to severe anemia and symptoms can worsen withfever or get worse when a patient is exposed to certain medicines,chemicals, or infectious agents. Blood transfusions are often needed.Such a patient generally has a greater risk of having a child with alphathalassemia major. (iv) Alpha thalassemia major occurs when four HBAgenes are missing. This causes severe anemia and in most cases a babywith this condition will die before birth.

Both alpha- and beta-forms of thalassemia major can cause significantcomplications. For example, people with thalassemia can get too muchiron in their bodies, either from the disease itself or from frequentblood transfusions. The iron overload can result in damage to the heart,liver and endocrine system which includes glands that produce hormonesthat regulate processes throughout the body. Bone deformities are alsocommon since thalassemia can make the bone expand, causing bone towiden. Thalassemia is also often accompanied by the destruction of alarge number of red blood cells, making the spleen to work harder thannormal and enlarge (splenomegaly). Splenomegaly can make anemia worseand reduce the life of transfused red blood cells. If the spleen growstoo big, it will have to be surgically removed. People with thalassemiamajor have an increased risk of infection. This is especially true whenthe spleen has been removed from the patient's body due to theaforementioned severe splenomegaly.

Infections are major complications for thalassemia patients andconstitute the second most common cause of mortality and morbidity forthese patients. Major causative organisms of bacterial infections inthalassemic patients include Klebsiella sp., Candida albicans,Staphylococcus aureus, Yersinia enterocolitica, Pseudomonas sp. andStreptococcus pneumoniae. For most of these bacteria, vaccines are notavailable. Where infection is suspected, the main causes to beconsidered include splenectomy, transmission of pathogens by bloodtransfusions, iron overload and iron chelation. As the body's immunesystem of a thalassemic patient is already sharply suppressed due to areduction in neutrophil numbers, it is crucial to reduce mortality byrecognizing and presumptively treating infections in a patient asquickly as possible. PCR-based methods facilitate the detection ofpathogenic DNA components in biological samples, see Vosberg, HumanGenetics 83(1):1-15, 198 which is incorporated reference). Treatment ofa subject identified or characterize as having, or at risk of having,thalassemia may include treatment of an infection with an antibiotic orother anti-microbial agent that kills or inhibits the growth of themicroorganisms described above or other infectious microbes in thesubject.

Treatment of alpha thalassemia may include: daily doses of folic acid;blood transfusions (as needed); surgery to remove the spleen; medicinesto reduce extra iron in the body (iron chelation therapy) such asDeferoxamine or Deferasirox); avoidance of oxidant drugs in hemoglobin Hdisease; transplantation of blood or bone marrow stem cells from ahealthy donor or with genetically modified homologous cells (genetherapy) correcting the defect in HBA levels. Genetic counseling mayalso be used as a preventative treatment to reduce the risk of offspringhaving or carrying alpha thalassemia or sickle cell disease. Typically,thalassemia major patients are treated by regular blood transfusions andchelation therapy. Individuals positive for the HBA12 gene (see FIGS.4B, 4C, 4F and 4H) and borderline HbA₂ are considered as betathalassemia carriers.

Sickle Cell Disease and Trait. Normally, humans have hemoglobin A, whichconsists of two alpha and two beta chains, hemoglobin A2, which consistsof two alpha and two delta chains, and hemoglobin F, consisting of twoalpha and two gamma chains in their bodies. Out of these three types,hemoglobin F dominates until about 6 weeks of age. Afterwards,hemoglobin A dominates throughout life. In people diagnosed with sicklecell disease, at least one of the β-globin subunits in hemoglobin A isreplaced with what's known as hemoglobin S. In sickle cell anemia, acommon form of sickle cell disease, hemoglobin S replaces both β-globinsubunits in the hemoglobin.

Sickle cell conditions have an autosomal recessive pattern ofinheritance from parents. The types of hemoglobin a person makes in thered blood cells depend on what hemoglobin genes are inherited from heror his parents. If one parent has sickle cell anemia and the other hassickle cell trait, then the child has a 50% chance of having sickle celldisease and a 50% chance of having sickle cell trait. When both parentshave sickle cell trait, a child has a 25% chance of sickle cell disease,25% do not carry any sickle cell alleles, and 50% have the heterozygouscondition.

Treatment of sickle cell disease and trait include any of the treatmentsdescribed by the Centers for Disease Control (CDC) at hypertext transferprotocol secure://worldwideweb.cdc.gov/ncbddd/sicklecell/treatments.html (incorporated byreference). Drug treatments include hydroxyurea, antibiotics, folicacid, vitamin E and NSAIDS, opioids and other analgesics.

Blood transfusions may be used to treat symptoms of SCD. Many Saudi SCDpatients are presently treated with hydroxyurea to reduce pain and bytherapeutic transfusions of red blood cells. In addition,transplantation of blood or bone marrow stem cells from a healthy donoror transfer of genetically modified homologous cells (gene therapy) thatproduce normal, non-Sickle cells. Genetic counseling may also be used asa preventative treatment to reduce the risk of offspring having orsickle cell disease.

Biological sample. A biological sample is a sample that contains anucleic acid detectable by PCR. A sample may contain genomic DNA as wellas RNA. Representative biological samples include whole blood, plasma,serum, buffy coat cells, and cells from a buccal swab. Other samples mayinclude spinal fluid, synovial fluid, lymph fluid, saliva, sputum,mucosal secretions, urine, and other biological fluid, cells, ortissues. In some embodiments a sample may contain cells obtained from anindividual that have been cultured in vitro or which have been frozen orotherwise preserved. In some embodiments, a biological sample willcontain a preservative, such as EDTA or a buffer or be isolated fromother components in an original biological sample, such as proteins. Thenucleic acid may be isolated from a cell or be isolated from anon-cellular material, such as cell-free DNA (cfDNA) from blood plasmaor serum. Preferably, a sample will be obtained non-invasively.

Samples may be obtained from normal subjects, subjects deemed at risk ofdeveloping an HBA associated disease, disorder or condition, or subjectsalready diagnosed with such a disease, disorder or condition. Samplesmay be taken from subjects for routine screening, from family members ofa patient, such as from a grandparent, parent, sibling or cousin, from aperson prior to marriage or prior to a pregnancy, from a pregnant womanor from a subject in utero.

Polymerase Chain Reaction (PCR). The present invention relates toprimers as well as primer pools comprising multiple primer pairs or aprimer pool that recognize, anneal to and simultaneously amplifytargeted HBA1, HBA2 and HBA12 genes. The practice of the presentinvention will employ, unless otherwise indicated, conventionaltechniques of molecular biology, microbiology and recombinant DNAtechniques, which are within the skill of the art including variousmultiplex PCR procedures including quantitative PCR. Such techniques areexplained fully in the literature and specific recipes and conditionsfor PCR reactions are provided in the examples; see, e.g., Sambrook,Fritsch & Maniatis, Molecular Cloning; A Laboratory Manual, SecondEdition, (1989) (hereinafter “Maniatis”); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Hames & S. J.Higgins, eds., 1984); A Practical Guide to Molecular Cloning (B. Perbal,1984); and a series, Methods in Enzymology (Academic Press, Inc.)—eachincorporated herein by reference in its entirety.

Other multiplex PCR methods are described and incorporated by referenceto: hypertext transfer protocolsecure://en.wikipedia.org/wiki/Multiplex_polymerase_chain_reaction (lastaccessed Nov. 12, 2018); Abbs, S; Bobrow, M (1992). “Analysis ofquantitative PCR for the diagnosis of deletion and duplication carriersin the dystrophin gene”. Journal of Medical Genetics. 29 (3): 191-96;Morlan, John; Baker, Joffre; Sinicropi, Dominick (2009). “MutationDetection by Real-Time PCR: A Simple, Robust and Highly SelectiveMethod”. PLoS ONE. 4 (2): e4584; hypertext transfer protocolsecure://worldwideweb.sciencedirect.com/topics/agricultural-and-biological-sciences/multiplex-polymerase-chain-reaction(last accessed Nov. 11, 2018); and Elnfro, et al., Clin Microbiol Rev.2000 October; 13(4): 559-570; hypertext transfer protocolsecure://worldwide web.ncbi.nlm.nih.gov/pmc/articles/PMC88949/(lastaccessed Nov. 11, 2018).

As used herein, the terms “polynucleotide” and “oligonucleotide” referto primers, probes, oligomer fragments to be detected, oligomer controlsand unlabeled blocking oligomers and shall be generic topolydeoxy-ribonucleotides (containing 2-deoxy-D-ribose), topolyribonucleotides (containing D-ribose), and to any other type ofpolynucleotide which is an N-glycoside of a purine or pyrimidine base,or modified purine or pyrimidine bases. There is no intended distinctionin length between the term “polynucleotide” and “oligonucleotide”, andthese terms will be used interchangeably. These terms refer only to theprimary structure of the molecule. Thus, these terms include double- andsingle-stranded DNA, as well as double- and single-stranded RNA.

The term “primer” may refer to more than one primer and refers to asingle-stranded oligonucleotide, whether occurring naturally, as in apurified restriction digest, or produced synthetically, which is capableof acting as a point of initiation of synthesis along a complementarystrand when placed under conditions in which synthesis of a primerextension product or amplicon, as used herein, which is complementary toa nucleic acid strand is catalyzed.

Such conditions include the presence of four differentdeoxyribonucleoside triphosphates and a polymerization-inducing agentsuch as a thermostable nucleotide polymerase, DNA polymerase or reversetranscriptase, in a suitable buffer (“buffer” includes substituentswhich are cofactors, or which affect pH, ionic strength, etc.), and at asuitable temperature. The primer is preferably single-stranded formaximum efficiency in amplification.

As used herein, the term “target sequence” refers to a region of thepolynucleotide which is to be amplified and/or detected, such assequences forming all or part of an HBA1, HBA2 or HBA12 gene. “Targetgene” refers to a target sequence wherein the region of thepolynucleotide is a full functional gene of an organism that codes for apolypeptide or for an RNA chain that has a function in the organism.

As used herein, the term “thermostable nucleotide polymerase” refers toan enzyme which is relatively stable to heat when compared to nucleotidepolymerases from E. coli and which catalyzes the polymerization ofnucleosides. Generally, the enzyme will initiate synthesis at the 3-endof the target sequence utilizing the primer, and will proceed in the3-direction along the template until synthesis terminates. Arepresentative thermostable enzyme isolated from Thermus aquaticus (Taq)is described in U.S. Pat. No. 4,889,818 and a method for using it isdescribed in Saiki et al., (1988), Science 239:487 (both incorporatedherein by reference in their entirety). In one advantageous embodimentthe multiplex PCR was performed by testing and selecting an annealingtemperature ranging from 65, 66, 67, 78, 69, to 70° C.; testing andselecting an annealing time ranging from 1, 1.25, 1.5, 1.75 to 2minutes. The quantity of primers was selected to range between 0.5, 1,1.5, 2, 2.5 to 3 μl per primer at a primer concentration of 10 μM andthe number of cycles were selected so as to provide for the properappearance of the amplicons. The reaction volume ranging from 25, 30,35, 40, 45 to 50 μl was selected.

In some embodiments, the primers disclosed herein may be used in aquantitative PCR method, such as a method that employs TaqMan hydrolysisprobes. Such methods are known and incorporated by reference tohypertext transfer protocol secure://en.wikipedia.org/wiki/TaqMan (lastaccessed Nov. 13, 2018) and hypertext transfer protocolsecure://en.wikipedia.org/wiki/Real-time_polymerase_chain_reaction (lastaccessed Nov. 13, 2018).

Primers developed by the inventors. The following ten primers may beused in the multiplex PCR based method of the invention, preferably,primers having SEQ ID NOS: 1-6 are used based on their high specificityand sensitivity for amplifying DNA encoding HBA12.

SEQ Name of ID NO: the primer Sequence (5′−>3′)  1 MA12FGCCCTCGGCCCCACTGACCCTCTT  2 MA2R CTCCCTGCAGTTCTCCCTCCCCAGC  3 MA1FTGTTTATTCCTTCCCGGTGCCTGTCACTCAA  4 MA12R AGGGTCAGTGGGGCCGAGGGCCCAGG  5MA2F TCTCCCCTGTCCTTTCCCTACCCAGAGC  6 MA2R CTCCCTGCAGTTCTCCCTCCCCAGC  7MA12aR AGGGTCAGTGGGGCCGAGGGCCCA  8 MA2SF GATTGGGCGAAGCCCTCCGGCTCG  9MA1R CCCAAGGGGCAAGAAGCATGGCCA 10 MA2SR TCAGTGCGGCCCAGGCCCGCAG

In some embodiments primers that amplify other genes associated withBA-related status may be included, for example, primers that amplify theARTX gene.

Modified primers. In some embodiments of the invention, PCR or otherprimer-based nucleic acid detection techniques may be performed with amodified primer, such as a primer of SEQ ID NO: 1-10 that is labeledwith a reporter fluorophone and/or a quencher molecule. Such reportersand quenchers are known in the art, see, for example, hypertext transferprotocol secure://worldwideweb.bio-rad.com/en-us/applications-technologies/introduction-pcr-primer-probe-chemistries?ID=LUSOJW3Q3(incorporated by reference, last accessed Oct. 30, 2018).

Primer modifications include, but are not limited to, substitution in anoligonucleotide of: 2-aminopurine for dA; 2,6-diaminopurine for dA;deoxyuridine (dU) for dT; 5-methyl dC for dC to increase Tm up to 0.5°C.; hydroxymethyl dC for dC; or deoxyinosine or 5-nitroindole as a“universal base” for any dA, dC, dG or dT in an oligonucleotide primer.Additional modifications include the incorporation or substitution of5-hydroxybutynl-2′-deoxyuridine which is a duplex stabilizing modifiedbase; incorporation or substitution of 8-aza-7-deazaguanosine thateliminates naturally occurring, non-Watson-and-Crick secondarystructures associated with guanine-rich sequences; substitution of alocked nucleic acid base which has a modification to its ribose backbonethat locks the base into a C3′-endo position, for one or morenucleotides in a primer. Other modifications include incorporation ofinverted dT at a 3′-end of an oligonucleotide to inhibit degradation by3′ exonucleases or extension by DNA polymerases; incorporation ofinverted dideoxy-T at a 5′ end of a sequence to prevent unwanted 5′ligations, or incorporation of dideoxy-C as a 3′ chain terminator. Othermodifications are incorporated by reference to hypertext transferprotocol secure://worldwideweb.idtdna.com/site/Catalog/Modifications/Category/7 (last accessed Nov.7, 2018) or the 3′, internal, or 5′ modifications described by andincorporated by reference to hypertext transfer protocolsecure://worldwideweb.thermofisher.com/us/en/home/life-science/oligonucleotides-primers-probes-genes/custom-dna-oligos/oligo-configuration-options.html#5prime(last accessed Nov. 7, 2018). One, two, three or more of thesemodifications may be incorporated into an oligonucleotide primersequence, such as those of SEQ ID NOS: 1-9, disclosed herein. A primermay also be modified by addition of a modified base, such as thosedescribed above at a 5′ or 3′ end. Advantageously, the method of theinvention may be performed efficiently, simply and at low cost usingunmodified primers.

Kits. Another aspect of the invention is a kit for determining HBAgenotype using the multiplex PCR methods described herein. Such a kitmay include one or more of a buccal swab, a blood collection device orother device for taking a biological sample, a PCR tube containing theprimers disclosed herein, such as primers having SEQ ID NOS: 1-10,dNTPs, a DNA polymerase, control templates, molecular weight standardssuch as ladders or allelic ladders, as well as equipment for performinga PCR. In other embodiments, the kit may contain the primers disclosedherein and reagents for quantitative PCR. Such kits may also containpackaging materials, envelopes or containers for shipping biologicalsamples, instructions for use and for communicating the test results,such as a web address or postal or shipping envelope.

Correlations with disease. The table below correlates particular patientstatus with HBA genotypes, for example, a normal subject may carry twocopies of α1 and two copies of α12. Other disease or trait correlationsmay be determined using the chart below. The multiplex PCR method of theinvention may be used to determine a person's genotype and thus provideuseful information about their status. Other correlations between α1, α2and α12 genotype and patient status are disclosed by and incorporated byreference to Borgio, Saudi Med J. 2015 November; 36(11): 1271-1276, see,for example, Table 1 and FIG. 2 of that reference.

Condition HBA Genotypes Normal a12/a1 a12/a1 Silent α-thal a12/— a12/a1Heterozygous  —/— α-thal trait a12/a1 Homozygous a12/— α-thal traita12/— HbH disease a12/—  —/— Hb Bart's  —/—  —/— Sickle Cell Sickle Celltrait

Correlations Between Multiplexing PCR Results and Sickle Cell Anemia andThalassemia.

Identification or characterization of a patient as having an a12/a1,a12/a1 genotype and as being in normal condition may trigger a decisionto provide genetic counselling classifying the subject as one withlittle or no risk of having a child with alpha-thalassemia, to removethe patient from a treatment for thalassemia, or to substitute adifferent treatment not based on misdiagnosis of thalassemia or SCD.Identification or characterization of a patient as having an a12/--,a12/a1 genotype and as having silent alpha thalassemia may trigger adecision to provide genetic counseling, such as premarital counseling,to explain the risks of a child having alpha thalassemia; or to removethe patient from a treatment for thalassemia.

Identification or characterization of a patient as having an --/--,a12/a1 genotype and as having heterozygous alpha thalassemia trait maytrigger a decision to provide genetic counseling, such as premaritalcounseling, to explain the risks of a child having alpha thalassemia; totreat the patient for mild anemia, or to differentially diagnose thepatient as having heterozygous alpha thalassemia which can be mistakenfor iron deficiency anemia or beta thalassemia. Treatments may includeadministration of folic acid, blood transfusions, bone marrowtransplantation, stem cell transplantation, gene therapy, splenectomy,surgery to remove gallstones and treatment of secondary complicationssuch as those resulting from a febrile episode or from infection with amicrobe. A drug treatment may include administering one or more drugssuch as Exjade, hydroxyurea, deferasirox, deferiprone, ferriprox, orJadenu, Treatment with an antibiotic may include administering one ormore antibiotics in the following classes of antibiotics: penicillins,tetracyclines, cephalosporins, quinolones, lincomycins, macrolides,sulfonamide, glycopeptides, aminoglycosides, or carbapenems. Treatmentof yeast infections may be made using a drug in the same classes asmiconazole, terconazole, or fluconazole.

Identification or characterization of a patient as having an a12/--,a12/--genotype and as having homozygous alpha thalassemia trait maytrigger a decision to provide genetic counseling, such as premaritalcounseling, to explain the risks of a child having alpha thalassemia; totreat the patient for mild anemia, or to differentially diagnose thepatient as having heterozygous alpha thalassemia which can be mistakenfor iron deficiency anemia or beta thalassemia. Treatments may includeadministration of folic acid, blood transfusions, bone marrowtransplantation, stem cell transplantation, gene therapy, splenectomy,surgery to remove gallstones and treatment of secondary complicationssuch as those resulting from a febrile episode or from infection with amicrobe. A drug treatment may include administering one or more drugssuch as Exjade, hydroxyurea, deferasirox, deferiprone, ferriprox, orJadenu, Treatment with an antibiotic may include administering one ormore antibiotics in the following classes of antibiotics: penicillins,tetracyclines, cephalosporins, quinolones, lincomycins, macrolides,sulfonamide, glycopeptides, aminoglycosides, or carbapenems. Treatmentof yeast infections may be made using a drug in the same classes asmiconazole, terconazole, or fluconazole.

Identification or characterization of a patient as having a12/--, --/--genotype and as having HbH disease may trigger a decision to providegenetic counseling, such as premarital counseling, to explain the risksof a child having alpha thalassemia, to treat the patient for HbHdisease, or to differentially diagnose the patient from other forms ofthalassemia or other conditions. Treatments may include administrationof folic acid, blood transfusions, bone marrow transplantation, stemcell transplantation, gene therapy, splenectomy, surgery to removegallstones and treatment of secondary complications such as thoseresulting from a febrile episode or from infection with a microbe. Adrug treatment may include administering one or more drugs such asExjade, hydroxyurea, deferasirox, deferiprone, ferriprox, or Jadenu,Treatment with an antibiotic may include administering one or moreantibiotics in the following classes of antibiotics: penicillins,tetracyclines, cephalosporins, quinolones, lincomycins, macrolides,sulfonamide, glycopeptides, aminoglycosides, or carbapenems. Treatmentof yeast infections may be made using a drug in the same classes asmiconazole, terconazole, or fluconazole.

Identification or characterization of a patient as having a --/--, --/--genotype and as having Bart's disease may trigger a decision to providegenetic counseling, such as premarital counseling, to explain the risksof a child having alpha thalassemia; to treat the patient HbH disease,or to differentially diagnose the patient from other forms ofthalassemia or other conditions. Treatments may include intrauterineblood transfusions including those performed at an early gestationalage, post-natal bone marrow transplantation or continued chronic bloodtransfusions, stem cell transplantation, or gene therapy. A drugtreatment may include administering one or more drugs such as Exjade,hydroxyurea, deferasirox, deferiprone, ferriprox, or Jadenu, Treatmentwith an antibiotic may include administering one or more antibiotics inthe following classes of antibiotics: penicillins, tetracyclines,cephalosporins, quinolones, lincomycins, macrolides, sulfonamide,glycopeptides, aminoglycosides, or carbapenems. Treatment of yeastinfections may be made using a drug in the same classes as miconazole,terconazole, or fluconazole.

Applications. Methods according to the invention may be employed todetermine whether a person has, is at risk of, is a carrier of sicklecell anemia or thalassemia, or other diseases, disorders, or conditionsassociated with a HBA genotype. It may be used for premarital orprenatal screening for disorders, diseases or conditions associated withabnormal expression of HBA1, HBA2 or with presence of HBA12. The methodmay also be used to assess the effects of genetic or pharmacologicaltreatment of genetic diseases associated with abnormal expression ofHBA1 or HBA2, or expression of HBA12, for example, after a bone marrowor other transplant of cells used to correct a genetic defect.

Multiplexing. The inventors designed many oligonucleotides for accuratePCR amplification of the HBA1, HBA2 and HBA12 genes. Oligonucleotideswere evaluated for their specificity and utility for single locus PCR.Sets of primers for the multiplex PCR reaction such as those shown byFIG. 2 were selected based on melting and annealing temperaturecalculations for single locus PCR. These primers range in length from18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 to 31 bp. The meltingtemperature (Tm) of the designed primers was calculated and the Tm ofeach primer was maintained in the range of 59, 60, 65, 70, to 75° C. orany intermediate temperature; Tm differences between members of a primerpair was maintained at no more than 1, 2, 3, 4, or 5° C., preferably nomore than 3° C.

The self-dimers or primer-dimers formations were estimated and theprimers with a high level of self-dimer or primer-dimer formations wereexcluded. Simultaneously, the GC and the AT ratio was maintained.

The inventors confirmed the efficacy of the primers in a single locusPCR for individual amplicons of the respective genes, after which, theinventors verified all the parameters to confirm the PCR components.

After the amplification of the single locus PCR for the individualamplification of the HBA1, HBA2 and HBA12 genes separately, the mostappropriate temperature for the PCR thermal profile were verified.Additionally, the most appropriate annealing temperature was testedusing various types of PCR, including gradient PCR reaction. Theprotocol for the multiplex PCR, including the PCR mix and the thermalprofiles, were adjusted to obtain the required number of PCR products.Various combinations of PCR mixtures, including bovine serum albumin,betaine and MgCl2 were tested to obtain the required PCR products in asingle reaction. Thermal cycling reactions were set at various levels tohave the three PCR amplicons in perfect form. The annealing temperatureof the multiplexing PCR was considered very carefully based on Tm. Theextension time was carefully considered to avoid the incomplete ampliconformation.

Reference guide and product confirmation. The HBA2, HBA1 and HBA12 geneshave been successfully amplified simultaneously in a single PCR tube(FIG. 3) and a quick reference guide for the multiplex PCR resultanalysis and interpretation of these specific genotypes has beendeveloped as shown by FIG. 4.

All the amplicons produced during the single locus PCR and the multiplexPCR were purified and confirmed by direct sequencing. All the PCRproducts, specifically the multiplexing products, have been gel elutedindividually from the gel and purified. All the PCR products weresequenced with the respective primers (FIGS. 5-8). The sequences werecompared for the origin of the amplicons and the expected region.

The inventors also confirmed that all the multiplex expectedband/amplicons (1874 bp, 1231 bp and 369 bp) were of accurate length andoriginated from the expected regions. Two non-specific amplicons betweenthe regions 1874 bp and 1231 bp, which were 1517 and 1550 bp in length,respectively (FIG. 3) were identified. These two non-specific ampliconswere not used in the interpretation of the multiplex results; rather theallelic ladder was used for comparison and interpretation.

EXAMPLES

The following examples illustrate various aspects of the presentinvention. They are not to be construed to limit the claims in anymanner whatsoever.

Example 1 Designing of Specific Primers for HBA1, HBA2 and HBA12

HBA1, HBA2 and HBA12 genes were sequenced and used to design thespecific primers for the amplification of these genes in samplesobtained from the Saudi population, see FIG. 1. The primers for theamplification of these three genes were designed specifically for eachgene. All the primers have been designed with the sequence length from24 to 31 bp nucleotides. All the manually designed primers weresubjected for the possible primer-primer dimer or interactions. NationalCenter for Biotechnology Information (NCBI) tool and Basic LocalAlignment Search Tool (PRIMER-BLAST) were used to identify thespecificity and the possible amplification of the non-specific amplicon.

Example 2 Single Locus Polymerase Chain Reaction

A simple PCR procedure for the amplification of individual alpha globingenes was standardized. Each set of primers was thoroughly checked forthe accurate amplification of specific amplicons without amplificationof any nonspecific amplicons. The inventors standardized the individualPCR with the annealing temperature ranging from 65-68° C. to make theprimers suitable for multiplexing.

The amplification of the individual PCR products was carried out atvolume of 25 μL of reaction: 1× Top Taq Buffer; 10 μM Reverse primer; 10μM Forward primer; Q-reagent; 25 mM dNTP; 2.5 U Top Taq DNA polymerase;and 25 ng/μl DNA Template. Temperature profile: 94° C.-5 mins; 35 cyclesof 94° C.-30 seconds, Annealing temperature 57-67/30 seconds; 72° C./0.5to 2 mins and final extension 72° C.-10 mins.

Example 3 Multiplex PCR

A pool of oligonucleotides (FIG. 2) was selected for the simultaneousmultiplex PCR amplification of HBA, HBA2 and HBA12 genes in a single PCRtube. The PCR recipe in a total volume 50 μl for the multiplexamplification of HBA1, HBA2 and HBA12 genes was PCR 10× Buffer-5.0 μl;Q-Reagent-10 μl; 10 mM dNTPs-1.0 μl; 2.5 U Top Taq DNA polymerase-0.3μl; 10 μM primers such as, Ma12F—0.5 μl; Ma2R—2.0 μl; Ma2F—2.0 μl;Ma12R—1.5 μl; Ma1F—1.5 μl; 25 ng/μl Sample DNA-2.0 μl and nuclease freedistilled water-24.2 μl.

The PCR thermal profile for the amplification for the HBA1, HBA2 andHBA12 genes in a single PCR tube is shown below.

Step 1: Initial denaturation at 95° C. for 3 mins.

Step 2: Denaturation at 95° C. for 30 secs.

Step 3: Annealing at 69° C. for 1 min. 30 secs.

Step 4: Extension at 72° C. for 2 mins. 15 secs.

Step 5: Repeat: Go to Step 2, 30 cycles.

Step 6: Final Extension at 72° C. for 5 mins.

Step 7: Store at 4° C.

The above protocol was verified using thermal cyclers from variousmanufacturers including those available from BioRad, Biometra andEppendorf.

Terminology. Terminology used herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

The headings (such as “Background” and “Summary”) and sub-headings usedherein are intended only for general organization of topics within thepresent invention, and are not intended to limit the disclosure of thepresent invention or any aspect thereof. In particular, subject matterdisclosed in the “Background” may include novel technology and may notconstitute a recitation of prior art. Subject matter disclosed in the“Summary” is not an exhaustive or complete disclosure of the entirescope of the technology or any embodiments thereof. Classification ordiscussion of a material within a section of this specification ashaving a particular utility is made for convenience, and no inferenceshould be drawn that the material must necessarily or solely function inaccordance with its classification herein when it is used in any givencomposition.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

It will be further understood that the terms “comprises” and/or“comprising,” when used in this specification, specify the presence ofstated features, steps, operations, elements, and/or components, but donot preclude the presence or addition of one or more other features,steps, operations, elements, components, and/or groups thereof.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items and may be abbreviated as“/”.

In some instances, the text available via the link on the “lastaccessed” date may be incorporated by reference.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “substantially”, “about” or“approximately,” even if the term does not expressly appear. The phrase“about” or “approximately” may be used when describing magnitude and/orposition to indicate that the value and/or position described is withina reasonable expected range of values and/or positions. For example, anumeric value may have a value that is +/−0.1% of the stated value (orrange of values), +/−1% of the stated value (or range of values), +/−2%of the stated value (or range of values), +/−5% of the stated value (orrange of values), +/−10% of the stated value (or range of values),+/−15% of the stated value (or range of values), +/−20% of the statedvalue (or range of values), etc. Any numerical range recited herein isintended to include all subranges subsumed therein.

Disclosure of values and ranges of values for specific parameters (suchas temperatures, molecular weights, weight percentages, etc.) are notexclusive of other values and ranges of values useful herein. It isenvisioned that two or more specific exemplified values for a givenparameter may define endpoints for a range of values that may be claimedfor the parameter. For example, if Parameter X is exemplified herein tohave value A and also exemplified to have value Z, it is envisioned thatparameter X may have a range of values from about A to about Z.Similarly, it is envisioned that disclosure of two or more ranges ofvalues for a parameter (whether such ranges are nested, overlapping ordistinct) subsume all possible combination of ranges for the value thatmight be claimed using endpoints of the disclosed ranges. For example,if parameter X is exemplified herein to have values in the range of 1-10it also describes subranges for Parameter X including 1-9, 1-8, 1-7,2-9, 2-8, 2-7, 3-9, 3-8, 3-7, 2-8, 3-7, 4-6, or 7-10, 8-10 or 9-10 asmere examples. A range encompasses its endpoints as well as valuesinside of an endpoint, for example, the range 0-5 includes 0, >0, 1, 2,3, 4, <5 and 5.

As used herein, the words “preferred” and “preferably” refer toembodiments of the technology that afford certain benefits, undercertain circumstances. However, other embodiments may also be preferred,under the same or other circumstances. Furthermore, the recitation ofone or more preferred embodiments does not imply that other embodimentsare not useful, and is not intended to exclude other embodiments fromthe scope of the technology. As referred to herein, all compositionalpercentages are by weight of the total composition, unless otherwisespecified. As used herein, the word “include,” and its variants, isintended to be non-limiting, such that recitation of items in a list isnot to the exclusion of other like items that may also be useful in thematerials, compositions, devices, and methods of this technology.Similarly, the terms “can” and “may” and their variants are intended tobe non-limiting, such that recitation that an embodiment can or maycomprise certain elements or features does not exclude other embodimentsof the present invention that do not contain those elements or features.

Although the terms “first” and “second” may be used herein to describevarious features/elements (including steps), these features/elementsshould not be limited by these terms, unless the context indicatesotherwise. These terms may be used to distinguish one feature/elementfrom another feature/element. Thus, a first feature/element discussedbelow could be termed a second feature/element, and similarly, a secondfeature/element discussed below could be termed a first feature/elementwithout departing from the teachings of the present invention.

The description and specific examples, while indicating embodiments ofthe technology, are intended for purposes of illustration only and arenot intended to limit the scope of the technology. Moreover, recitationof multiple embodiments having stated features is not intended toexclude other embodiments having additional features, or otherembodiments incorporating different combinations of the stated features.Specific examples are provided for illustrative purposes of how to makeand use the compositions and methods of this technology and, unlessexplicitly stated otherwise, are not intended to be a representationthat given embodiments of this technology have, or have not, been madeor tested.

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference,especially referenced is disclosure appearing in the same sentence,paragraph, page or section of the specification in which theincorporation by reference appears.

The citation of references herein does not constitute an admission thatthose references are prior art or have any relevance to thepatentability of the technology disclosed herein. Any discussion of thecontent of references cited is intended merely to provide a generalsummary of assertions made by the authors of the references, and doesnot constitute an admission as to the accuracy of the content of suchreferences.

The invention claimed is:
 1. A method for treating a subject at risk ofa hemoglobin A (HBA) associated disease or trait by detecting Hemoglobinalpha 1 (HBA1) gene, Hemoglobin alpha 2 (HBA2) gene, and Hemoglobinalpha 12 (HBA12) genes in the subject, comprising: obtaining a samplefrom the subject; contacting the sample with at least one set of primersfor each of HBA1, HBA2 and HBA12 under conditions suitable for theprimer sets to amplify DNA in the sample by multiplex polymerase chainreaction (PCR), wherein the set of HBA1 primers are MA1F (SEQ ID NO: 3)and MA12R (SEQ ID NO: 4), the set of HBA2 primers are MA2F (SEQ ID NO:5) and MA2R (SEQ ID NO: 2 or 6) and the set of HBA12 primers are MA12F(SEQ ID NO: 1) and MA2R (SEQ ID NO: 2 or 6), detecting the presence of,or levels of, HBA1, HBA2 and HBA12 DNA amplified by each primer set,thereby detecting a genotype of the subject, and treating a course of anHBA-associated disease or treating a trait using a detected genotype incombination with clinical data to inform a treatment decision.
 2. Themethod of claim 1, wherein said subject carries gene conversion HBA12.3. The method of claim 1, wherein said subject does not carry geneconversion HBA12.
 4. The method of claim 1, wherein the sample is blood,plasma or serum, or white blood cells, or cells obtained from a buccalswab.
 5. The method of claim 1, wherein the sample is cells obtainedfrom a buccal swab.
 6. The method of claim 1, wherein the primers forHBA1, HBA2 and HBA12 are part of a single primer pool.
 7. The method ofclaim 1, further comprising identifying the subject as being at risk ofsickle cell disease when the presence of amplified HBA1, HBA2 and HBA12is detected.
 8. The method of claim 1, further comprising identifyingthe subject as having sickle cell trait or sickle cell disease when thepresence of amplified HBA1, HBA2 and HBA12 is detected.
 9. The method ofclaim 1, further comprising identifying the subject as being at risk ofsickle cell disease when the presence of amplified HBA1, HBA2 and HBA12is detected and treating the subject for sickle cell disease with atleast one of penicillin, hydroxyurea, a transfusion of red blood cells,or by hematopoietic stem cell transplantation.
 10. The method of claim1, further comprising identifying the subject as being at risk ofthalassemia when the presence of amplified HBA1, HBA2 and HBA12 isdetected.
 11. The method of claim 1, further comprising identifying thesubject as being at risk of thalassemia when the presence of amplifiedHBA1, HBA2 and HBA12 is detected and treating the subject forthalassemia with at least one of a blood transfusion, iron chelationtherapy, or folic acid.
 12. A method for genetic counselling comprisingthe method of claim 1, wherein said treating a course of anHBA-associated disease or trait using the detected genotype incombination with clinical data comprises informing the subject of his orher HBA-associated gene associations.
 13. The method of claim 12,wherein said genetic counseling is premarital counseling.
 14. The methodof claim 12, wherein said subject is identified as having an a12/a1,a12/a1 genotype.
 15. The method of claim 12, wherein said subject isidentified as having an a12/a1, a12/-- genotype.
 16. The method of claim12, wherein said subject is identified as having an a12/--, a12/--genotype.
 17. The method of claim 12, wherein said subject is identifiedas having an a12/--, --/-- genotype or a --/--, --/-- genotype.
 18. Amethod for differential diagnosis comprising the method of claim 1,wherein said treating a course of an HBA-associated disease or traitusing the detected genotype in combination with clinical data comprisesinforming a health care provider of the subject's HBA-associated geneassociations with sickle cell disease or with thalassemia.
 19. Themethod of claim 18, wherein said subject carries gene conversion HBA12.20. The method of claim 18, wherein said subject does not carry geneconversion HBA12.